Although acidulants have various functions in food processing and products, their principal role is to adjust the pH of foods, primarily to enhance and to modify flavor, but also to preserve foods. Acidulants are used to add sourness and a desired tartness to many food products. Because of the low pH created when used, food acids can also prevent growth of microorganisms that might cause food to spoil or cause food poisoning or diseases. Through chelation of trace metal ions and/or low pH, acidulants also prevent rancidity and discoloration of foods by functioning as synergists to antioxidants such as BHA, BHT, and ascorbic acid. They also are used as buffers during various stages of food processing as well as in finished products. Food acidulants may also be used for purposes other than imparting flavor or aiding in preservation. They function as melting modifiers for cheese spreads and mixtures used in making hard candy, as gelling agents, and as viscosity modifiers for doughs. The versatility of acidulants will certainly be a factor in their general increased use as food additives in the future.
U.S. markets for food acidulants include carbonated beverages, powdered soft-drink mixes, noncarbonated beverages (e.g., fruit juice drinks), flavored gelatin desserts, jams and jellies, candies, canned fruits and vegetables, pie fillings, yogurts, starch puddings, and wines. Beverages--including liquids and powders--are the largest end use for acids in food products. Acids are used in soft drinks to provide tartness and to modify sweetness, and they are used in canning tomatoes to alter pH in order to optimize heat processing. Properties of the various acids are important to consider for the different applications; in some uses, high solubility is important (e.g., for flavor concentrates), whereas in other uses sourness and speed of solution may be the critical factors (e.g., powdered beverage mixes).
Of all acids used in foods, citric acid is the most widely used. Malic acid generally is recognized as being versatile and a potential competitor with citric acid. Used as a food acidulant in the United States for only about fifteen years, malic acid has made its greatest inroads when new products are developed and when old products need improvement. It has been successful in replacing citric acid, to some degree, in some major uses, such as dry powder mixes and candies. In new products, it is sometimes used in combination with citric acid.
Beverages based on malic acid use 8 to 12% less acidulant by weight, on the average, than beverages using citric acid, with extremes of 5 to 22% less acidulant having been reported. Although the level of acid required for replacement varies with the type of flavor, level of sweetness, and level of carbonation used, there remains a clear economic incentive for replacing citric acid by malic acid.
Malic acid has penetrated some of citric acid's uses in the nectar and diet-drink market; some sugar-free and low-calorie soft-drink producers have switched to malic from citric acid because malic acid masks the off-taste produced by sugar substitutes (e.g., saccharin). Malic acid is used to enhance the flavor in fruit-flavored (especially the berry flavors) carbonated beverages and cream sodas. It is used in beverages by itself and, in some instances, in combination with citric acid. Newly marketed food and beverage products have been (and are expected to continue to be) malic acid's major acidulant growth market. Other carbonated and still beverages, candies, dessert powders, instant tea, syrups, and preserves are targets for growth. It is believed that malic acid's use in fruit drinks, particularly apple and berry flavors, will increase because of the apparent change in consumer preference for beverage flavors; a faster growth rate in consumption of non-cola-flavored beverages compared to cola beverages is foreseen.
Domestic demand for malic acid as a food and beverage acidulant has increased from about 4 million pounds in 1967 to about 11 million pounds in 1979. Domestic production in 1979 was over 15 million pounds, with the difference between production and demand being exported. The average annual growth rate for malic acid has been estimated between 3 and 8% per year.
Malic acid is naturally found in many fruits, such as apples, and is there produced not as a racemate but as L-malic acid. Regulations in most of Western Europe, with the exception of the United Kingdom, Norway, and Denmark, do not permit additives of a synthetic origin in food and beverages. Therefore, racemic malic acid is not allowed in food and beverages except in those countries mentioned. Consequently, there is a great economic incentive for a method of producing L-malic acid relatively inexpensively.
Presently, malic acid is made by hydration of maleic anhydride to afford racemic malic acid. Resolution of the racemate to obtain L-malic acid is expensive, hence its supply remains limited. The demand for L-malic acid remains high, and for the aforementioned reasons the use of malic acid as an acidulant, especially in Europe, undoubtedly would increase were L-malic acid economically competitive with citric acid.
Fumarase is an enzyme which catalyzes the interconversion of fumaric and L-malic acids under mild conditions typical of enzymatic reactions. The invention herein is based on our discovering a bacterial strain which produces a fumarase which can be easily purified, which has substantial thermal stability even at 50.degree. C., which converts fumaric acid to L-malic acid without any other detectable coproducts, and which can be efficiently immobilized. Our discovery thus makes possible a commercially feasible process of making L-malic acid.
In one aspect our invention is a biologically pure culture of a strain of Paracoccus denitrificans, FUM-14, NRRL B-15710. In another aspect our invention is a method of producing a fumarase by growing FUM-14 aerobically in a medium containing an assimilable source of carbon, nitrogen, and mineral nutrients, at a temperature from about 20.degree. to about 45.degree. C., and recovering the fumarase produced thereby. In still another aspect our invention is the fumarase produced by Paracoccus denitrificans when grown under the aforesaid conditions.